Confirmation of NF-Y binding to the proximal promoter region of the AKR1C1 gene. NF-Y is composed of three subunits NF-YA, NF-YB and NF-YC that are required for interaction with the CCAAT pentanucleotide sequence in the AKR1C1 gene promoter. Nuclear extracts (8µg) from (A) 2008, (B) 2008/C13*, (C) H23, (D) A549, and (E) HepG2 were incubated with WT probe (−120 to −91) from AKR1C1 gene along with 2µg of polyclonal antibody directed towards NF-YA, NF-YB or NF-Y(NF-YA+NF-YB+NF-YC). The negative control consisted of rabbit pre-immune control IgG (C-IgG). The formation of DNA-protein complexs is designated by solid arrows and the loss of protein-DNA complex’s and/or gel super-shift in the presence of the antibody is denoted by an asterisk. Each of the experiments was repeated at least three times with similar results.